Eur J Plant Pathol (2018) 151:427–438
https://doi.org/10.1007/s10658-017-1384-5
Bacterial canker of cherry trees, Prunus avium, in South
Africa
M. Otto & Y. Petersen & J. Roux & J. Wright &
T. A. Coutinho
Accepted: 7 November 2017 / Published online: 25 November 2017
# Koninklijke Nederlandse Planteziektenkundige Vereniging 2017
Abstract In the 1980’s the causal agents of bacterial
canker of cherry trees in South Africa was reported to be
Pseudomonas syringae pv. syringae and Pseudomonas
syringae pv. morsprunorum. Subsequently, no further
studies were undertaken on the disease or causal agents.
The aim of the current study was to conduct field sur-
veys to determine the current situation pertaining to
bacterial canker in the major cherry producing areas of
South Africa. Following isolations from infected trees,
strains were characterized using biochemical as well as
multilocus sequence analyses (MLSA). Pathogenicity
tests were undertaken with immature cherry fruit as well
as three different cherry cultivars. Although symptoms
of bacterial canker were present in all areas surveyed,
P. syringae isolates were only isolated from three sites in
the Western Cape Province. The isolates collected in this
M. Otto : T. A. Coutinho (*)
Department of Microbiology and Plant Pathology, Centre for
Microbial Ecology and Genomics, Forestry and Agricultural
Biotechnology Institute, University of Pretoria, Private Bag X20,
Pretoria 00282, South Africa
e-mail: teresa.coutinho@fabi.up.ac.za
Y. Petersen
Agricultural Research Council, Infruitec-Nietvoorbij, Private Bag
X5026, Stellenbosch 7599, South Africa
J. Roux
Department of Plant and Soil Sciences, Forestry and Agricultural
Biotechnology Institute, University of Pretoria, Private Bag X20,
Pretoria 0028, South Africa
J. Wright
574 Molly Ryde Street, Garsfontein, Pretoria 0181, South Africa
study showed a hypersensitive response on tobacco
leaves and were pathogenic on immature cherry fruit
and cherry trees. The phenotypic tests and MLSA using
four genes (cts, gapA, gyrB and rpoD) showed pheno-
typic and genetic identity with Pseudomonas syringae
pv. syringae. Selected strains induced a hypersensitive
response on tobacco leaves and were pathogenic on
immature cherry fruit and green cherry tree shoots.
The current study shows that P. syringae pv. syringae
is responsible for bacterial canker in the Western Cape
Province, South Africa.
Keywords Pseudomonas syringae . MLSA . Prunus .
Stone fruit
Introduction
Cherry trees (Prunus avium) are native to the area
between the Black and Caspian seas of Asia Minor,
and are commercially grown in many countries
(Alonso 2011; Lim 2012). In South Africa, the first
orchard, Nooitgedacht (=Gydo), was established in
1890 in the Koue Bokkeveld in the Western Cape
Province (Watson 2016). By 1939, 32 ha of cherry
trees had been planted and today Nooigedacht is still
one of the major cherry producing farms in the region.
Cherry trees are also grown commercially in three
other provinces, namely, Free State, Mpumalanga
and the North-West. In the 2012/13 and 2013/14 sea-
sons the Western Cape Province accounted for 74% of
the cherry production in South Africa [ca. 691
428
Megaton (MT) and 499 MT, respectively] followed by
the Free State with 23% and 16% in Mpumalanga (ca.
105 MT and 51 MT, respectively) (Potelwa and
Ntombela 2015).
As with other agricultural crops, pests and diseases
pose serious threats to the production of cherries. One
such disease is bacterial canker, which was first re-
ported to occur in South Africa on stone fruit trees,
including cherry trees, in 1953 (Doidge et al. 1953).
The disease is characterized by the formation of can-
kers on stems and branches, blossom blast, systemic
wilting and die-back of branches or the entire tree
(Agrios 2005). The first intensive studies on the dis-
ease in South Africa were undertaken in the 1980s
(Roos and Hattingh 1986, 1987a, b). Thereafter, no
further research was undertaken and the disease was
considered to be of little economic importance. Infect-
ed trees bearing poor yields were removed and
replaced.
In the 1980’s, the causal agents of bacterial canker
of cherry trees in South Africa were identified as
Pseudomonas syringae pv. syringae (Pss) and Ps.
syringae pv. morsprunorum race 1 (Psm1) and 2
(Psm2) (Roos and Hattingh 1986). Psm2 was consid-
ered to be the major pathogen causing bacterial canker
in the Free State Province while both pathovars were
well established in the Western Cape (Roos and
Hattingh 1986). P. syringae pv. avii (Psa) has only
been isolated from wild cherry in Europe (Ménard
et al. 2003); whilst a new bacterial species, Pseudo-
monas cerasi causing symptoms similar to bacterial
canker was recently identified as a pathogen of culti-
vated cherry trees (Kałużna et al. 2016a, b). Disease
outbreaks caused by Pss, Psm1 and Psm2 have been
reported globally (reviewed by Lamichhane et al.
2014), but P. cerasi has only been isolated in Poland
(Kałużna et al. 2016a). Pss has a broad host range and
is pathogenic to pome fruit as well as several cultivat-
ed stone fruits including cherry, plum, peach and
apricot (Kałużna et al. 2010a; Karimi-Kurdistani and
Harighi 2008; reviewed by Lamichhane et al. 2014;
Wenneker et al. 2013). The other P. syringae
pathovars have much narrower host ranges: Psm1
has primarily been isolated from cherry, plum and
apricot whereas Psm2 and P. cerasi have only been
associated with cherry (reviewed by Bultreys and
Kaluzna 2010; Kałużna et al. 2016a).
Eur J Plant Pathol (2018) 151:427–438
Currently, a combination of phenotypic, molecular
and physiological methods are used for the character-
ization of P. syringae pathogenic to cherry (Kałużna
and Sobiczewski 2009; Vicente et al. 2004). All
P. syringae pathovars pathogenic to cherry except for
Pseudomonas syringae pv. avii (Psa) produce a fluo-
rescent pigment which is visible under UV light (King
et al. 1954) and are Gram negative when tested with
3% KOH (Suslow et al. 1982). Phenotypic testing
methods including the LOPAT (Levan production,
Oxidase test, Potato soft rot, Arginine dihydrolase,
Tobacco hypersensitive reaction) (Lelliott et al.
1966), carbon source utilization tests (Young and
Triggs 1994), L-lactate utilization and GATTa tests
enable the identification of P. syringae pathovars and
races (Latorre and Jones 1979; Lelliott and Stead
1987). The virulence of P. syringae on cherry can be
assessed by inoculating these strains into susceptible
cherry tree organs such as immature fruitlets (Kałużna
and Sobiczewski 2009). Phenotypic methods are used
in combination with molecular methods in a polypha-
sic approach for the identification of P. syringae
pathovars and races from cherry. A commonly used
molecular tool to delineate phylogenetic relationships
within species or genera is Multi Locus Sequence
Analyses (MLSA). MLSA studies have been used to
delineate the P. syringae strains within the P. syringae
complex into 13 phylogroups (Berge et al. 2014;
Parkinson et al. 2011) corresponding to the
genomospecies derived from DNA:DNA hybridiza-
tion analyses (Gardan et al. 1999). For MLSA analy-
ses of P. syringae pathovars and races from cherry, the
housekeeping genes cts, gapA, gyrB, rpoD (Kałużna
et al. 2010a) and rpoB (Ait Tayeb et al. 2005) are often
sequenced.
Over the past few years South Africa has and is
still experiencing a severe drought and unusually
high temperatures. These abiotic factors have led to
increased reports of bacterial canker in stone fruit tree
orchards, including cherry orchards. In spring 2014,
trees in several cherry orchards in South Africa
showed typical symptoms of bacterial canker includ-
ing stem and branch cankers, die-back of branches
and leaf spots. The aim of this study was to conduct a
survey in cherry production areas of South Africa to
determine the prevalence of the disease and identify
the bacteria associated with the disease symptoms.
Eur J Plant Pathol (2018) 151:427–438
These results would then be compared to those ob-
tained in the 1980s when identification of bacteria
was based solely on chemotaxonomic criteria.
Materials and methods
Surveillance and sampling
Surveys were conducted in 11 sweet cherry orchards in
four provinces (Gauteng, Mpumalanga, Free State and
Western Cape) of South Africa in October/November
2014 and 2015. In each orchard, 10% of each cultivar
was randomly surveyed for the presence of bacterial
disease symptoms. Symptoms were noted as being pres-
ent or absent. Leaves, stem- and branch tissue showing
typical symptoms of bacterial canker were collected.
Diseased material was cut from trees using sterile equip-
ment, placed into polythene bags and kept at 4 °C until
analyses were undertaken.
Bacterial isolation and growth conditions
After surface sterilization with 70% ethanol, small
pieces of tissue (±3 mm) showing symptoms of bacterial
canker were cut from the leading edges of stem- and
branch cankers and/or leaf spots, and placed on to
nutrient agar (Merck, Darmstadt, Germany). Isolation
plates were incubated for 48 h at 28 °C. Pure cultures of
bacterial isolates were obtained by streaking selected
colonies onto sterile nutrient agar plates and incubated
for 48 h at 28 °C. Pure cultures were streaked onto
Kings B medium [20 g Difco proteose peptone No. 3
(Merck), 15 ml glycerol, 1.5 g K2HPO4, 1.5 g MgSO4 x
7H2O, 15 g agar and 1 L H2O] (King et al. 1954) and
incubated for 24 h at 28 °C. Strains where examined for
fluorescent pigment production under UV light (King
et al. 1954) and a Gram reaction was performed with
3%KOH (Suslow et al. 1982). All bacterial isolates were
tested for pathogenicity on bean pods (Phaseolus
vulgaris) as described by Latorre and Jones (1979)
followed by the testing of fluorescent Pseudomonas
isolates in triplicate on immature cherry fruit (suscepti-
ble cultivar ‘Bing’). Immature cherry fruit were surface
sterilized with 70% ethanol and left to air dry. Twenty-
four hour-old cultures of the Pseudomonas strains were
grown on King’s B medium and colonies were
429
suspended in sterile distilled water and the concentra-
tions were adjusted to approx. 108 CFU/ml (OD600 =
0.3). The sterilized immature cherry fruit were injected
with 20 μl of bacterial suspension using a hypodermic
syringe needle. Immature cherry fruit were treated with
sterile water to serve as a negative control. The inocu-
lated fruits were incubated for 96 h at 24 °C to assess
pathogenicity and the necrotic lesions that formed were
measured from the needle wounds to the widest edge of
the necrotic lesions. The pathogenicity of the strains was
rated according to the following scale modified from
Kałużna and Sobiczewski (2009): non-pathogenic
strains = 0–3 mm, moderately pathogenic = 3–6 mm
and highly pathogenic= >6 mm. The pathogenicity trial
on the immature cherry fruit was repeated. Of all bacte-
rial isolates, only 31 bacterial isolates belonging to
Pseudomonas gave a positive HR result on bean pods
(Phaseolus vularis) an were moderately pathogenic to
immature Bing fruit (Fig. 1). Brown tissue necrosis
developed at the site of infiltration and extended be-
tween four and six millimetre in diameter. No symptoms
developed in the fruit treated with sterile water (Fig. 2).
Bacterial identification
Phenotypic testing
Candidate pathogenic Pseudomonas isolates obtained
from the current study were further characterized using
phenotypic tests commonly used to identify P. syringae
strains. The available LOPAT and GATTa phenotypic
profile from P. syringae reference strains including Pss
LMG 1247T, Psm1 LMG 2222, Psm2 CFBP 3800 and
P. cerasi LMG 28609T was used for comparative
Fig. 1 Pathogenicity trials with Pseudomonas syringae pv.
syringae strains on Bing immature cherry fruit. All strains were
moderately pathogenic, causing between four and six millimetres
of tissue necrosis around inoculation site
430
Fig. 2 Pathogenicity trials with Pseudomonas syringae pv.
syringae strains on Bing immature cherry fruit. No symptoms
developed on control fruit treated with sterile water
purposes (Kałużna et al. 2010b, 2016a). LOPAT tests
including the production of levan (L), oxidase activity
(O), pectolytic activity on potato slices (P), arginine
utilization (A) and production of a hypersensitive reac-
tion (HR) on tobacco (T) (Nicotiana tabacum) were
determined for each bacterial strain as described by
Lelliott et al. (1966). Utilization of carbon sources in-
cluding erythritol, inositol, sorbitol, sucrose and manni-
tol was performed according to Young and Triggs
(1994). To differentiate between P. syringae pathovars
syringae and morsprunorum, GATTa tests were carried
out with all isolates and gelatine hydrolysis (G); aesculin
hydrolysis (A); tyrosinase activity (T) and utilization of
tartaric acid (Ta) were measured as described by
Goszczynska et al. (2000).
MLSA analyses
31 bacterial isolates characterized as Pss by biochem-
ical testing, all of which were isolated from three
farms (farm A, B and C) in the Western Cape
Table 1 Multilocus sequencing analyses (MLSA) primers used in this study to amplify housekeeping genes from bacterial strains isolated
from cherry trees with symptoms of bacterial canker in South Africa
Primers Sequence
cts-p 5′-GCCTCBTGCGAGTCGAAGATCACC −3’
cts-s 5′-CGAAGATCACGG TGAACATGCTGG-3’
gapA-p 5′-CCGGCSGARCTGCCSTGG-3’
gapA-s 5′-TCGARTGCACSGGBCTSTTCACC-3’
gyrB-p 5′-TCBGCRGCVGARGTSATCATGAC-3’
gyrB-s 5′-TTGTCYTTGGTCTGSGAGCTGAA-3’
rpoD-p 5′-AAGGCGARATCGAAATCGCCAAGCG-3’
rpoD-s 5′-GGAACWKGCGCAGGAAGTCGGCACG −3’
Eur J Plant Pathol (2018) 151:427–438
Province, were subjected to MLSA analyses. Geno-
mic DNA of the bacterial strains was extracted using
the Zymo Research Quick gDNA™ MiniPrep DNA
extraction kit (Inqaba Biotech, South Africa). Purified
DNA was quantified using a NanoDrop 2000 (Thermo
Fischer Scientific, Waltham, Massachusetts, USA).
The DNA was stored at −20 °C for further analysis.
Four housekeeping genes including cts (citrate syn-
thase), rpoD (sigma factor), gyrB (DNA gyrase B) and
gapA (glyceraldehyde-3-phosphate) were amplified
using primers designed by Hwang et al. (2005) and
Sarkar and Guttman (2004) (Table 1). PCR amplifica-
tion was performed as previously described by Morris
et al. (2008). All PCR products were visualized on 2%
agarose gels at 90 V for 30 min, and purified by using
ExoSAP PCR clean-up reagent as described by the
manufacturer (Affymetrix, Santa Clara, CA, USA).
Sequencing of the PCR amplification products was
performed as described by Yan et al. (2008) by Inqaba
Biotechnology (Pretoria, South Africa). All partial
sequences of the P. syringae strains used for MLSA
analyses were deposited into the National Centre of
Biotechnology Information (NCBI). GenBank data-
base, and accession numbers are available for four
DNA regions sequenced for each strain (Table 2).
Phylogenetic analyses
For MLSA analyses, reference sequences of other
P. syringae strains belonging to various pathovars and
PG’s were obtained from the Plant Associated and En-
vironmental Microbes database (PAMDB) (Almeida
et al. 2010) and the NCBI database (https://www.ncbi.
nlm.nih.gov/were) for comparative purposes with the
Reference
Hwang et al. 2005
Hwang et al. 2005
Hwang et al. 2005
Hwang et al. 2005
Hwang et al. 2005
Hwang et al. 2005
Sarkar and Guttman 2004
Sarkar and Guttman 2004
Eur J Plant Pathol (2018) 151:427–438 431

Table 2 GenBank accession numbers of the DNA sequences of cherry trees in three farms (farm A, B and C) located in the
four housekeeping genes amplified from Pseudomonas strains and Western Cape Province of South Africa
used for phylogenetic analysis of bacteria isolated from diseased

Location Strain number cts gapA gyrB rpoD

Farm A 132 KY594935 KY594967 KY594999 KY595031

154 KY594936 KY594968 KY595000 KY595032
166 KY594937 KY594969 KY595001 KY595033
168 KY594938 KY594970 KY595002 KY595034
181 KY594939 KY594971 KY595003 KY595035
Farm B 190 KY594940 KY594972 KY595004 KY595036
201 KY594941 KY594973 KY595005 KY595037
203 KY594942 KY594974 KY595006 KY595038
215 KY594943 KY594975 KY595007 KY595039
216 KY594944 KY594976 KY595008 KY595040
245 KY594945 KY594977 KY595009 KY595041
260 KY594946 KY594978 KY595010 KY595042
301 KY594947 KY594979 KY595011 KY595043
305 KY594948 KY594980 KY595012 KY595044
Farm C 38 KY594918 KY594950 KY594982 KY595014
41 KY594919 KY594951 KY594983 KY595015
42 KY594920 KY594952 KY594984 KY595016
48 KY594921 KY594953 KY594985 KY595017
52 KY594922 KY594954 KY594986 KY595018
53 KY594923 KY594955 KY594987 KY595019
56 KY594924 KY594956 KY594988 KY595020
66 KY594925 KY594957 KY594989 KY595021
68 KY594926 KY594958 KY594990 KY595022
72 KY594927 KY594959 KY594991 KY595023
78 KY594928 KY594960 KY594992 KY595024
81 KY594929 KY594961 KY594993 KY595025
82 KY594930 KY594962 KY594994 KY595026
83 KY594931 KY594963 KY594995 KY595027
101 KY594932 KY594964 KY594996 KY595028
126 KY594933 KY594965 KY594997 KY595029
127 KY594934 KY594966 KY594998 KY595030

bacterial strains obtained in the current study (Table 3).
The dataset was aligned using the MAFFT (Version 7)
online alignment tool (Katoh and Standley 2013) and
trimmed at both ends using BioEdit Sequence Alignment
Editor (Hall 1999). Partition-homogeneity tests were per-
formed in PAUP 4.0b10 (Swofford 2002) to establish if
the four genes could be combined to form a single
concatenated data set. JModelTest (Posada 2008) was
applied to all four data sets, as well as the concatenated
data set, to determine the best-fit evolutionary model to
apply to each gene and the concatenated dataset. The
model score was evaluated with the hierarchical likeli-
hood ratio (hLRT) and the standard Akaike Information
criterion (AIC). Neighbour-joining (NJ) trees were drawn
for each gene and the concatenated data by using MEGA
(Version 6) and PhyML (Version 3.1) respectively. Boot-
strap analysis with 1000 replicates was performed on all
five trees to assess the reliability of the clusters generated.
432 Eur J Plant Pathol (2018) 151:427–438

Table 3 Pseudomonas reference strains used for multilocus se- (PAMDB) (Almeida et al. 2010) and from reference strains in
quence analysis (MLSA). All the sequences were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/were)
the Plant Associated and Environmental Microbes database

Strain number Strain name Host Phylogroup Database

CFBP 3846PT Pseudomonas syringae pv. avii Prunus avium (cherry) 1 NCBI
CFBP 2212PT Pseudomonas syringae pv. tomato Solanum lycopersicum (tomato) 1 PAMDB
CFBP 2351PT Pseudomonas syringae pv. morsprunorum Prunus domestica (plum) 1 NCBI
MAFF 302280 Pseudomonas syringae pv. morsprunorum Prunus domestia (plum) 1 NCBI
BPIC 631 T Pseudomonas avellanae Corylus avellana (hazel) 1 NCBI
LMG 3487 Pseudomonas syringae pv. avellanae Corylus avellana (hazel) 1 NCBI
HRI-W 7872 Pseudomonas syringae pv. syringae Prunus domestica (plum cv. Opal) 2 NCBI
LMG 1247T Pseudomonas syringae pv. syringae Syringa vulgaris (lilac) 2 PAMDB
58 T Pseudomonas cerasi Prunus avium (cherry) 2 NCBI
Cit7 Pseudomonas syringae Citrus sinensis (navel orange) 2 PAMDB
M301765 Pseudomonas syringae pv. glycinea Glycine max (soybean) 3 PAMDB
B076 Pseudomonas syringae pv. glycinea Glycine max (soybean) 3 NCBI
R4_A29–2 Pseudomonas syringae pv. glycinea Glycine max (soybean) 3 PAMDB
CFBP 5067 Pseudomonas syringae pv. nerii Nerium oleander (oleander) 3 NCBI
NCPPB 3335 Pseudomonas syringae pv. savastanoi Olea europea (olive tree) 3 NCBI
ATCC 11528 Pseudomonas syringae pv. tabaci Nicotiana tabacum (tobacco) 3 NCBI
6605 Pseudomonas syringae pv. tabaci Nicotiana tabacum (tobacco) 3 NCBI
CFBP 4389T Pseudomonas palleroniana Oryza sativa (rice) Outgroup NCBI
CFBP 2022T Pseudomonas salomonii Allium sativum (garlic) Outgroup NCBI
DSM18862 Pseudomonas azotoformans Oryza sativa (rice) Outgroup NCBI

Pathogenicity testing on P. avium shoots
Pathogenicity trials on P. avium shoots were con-
ducted as previously described by Latorre and Jones
(1979), Roos and Hattingh (1983) and Roos (1986).
The vegetative shoots on two-year-old cherry trees
of three different cultivars (Bing, Giant Heidelfinger
and Stella) grown under field conditions were inoc-
ulated with three pathogenic strains of Pseudomo-
nas, as established from the immature cherry fruit
inoculations. The strains used were Pss 38, 190 and
154 and were representative of three different farms
surveyed (farm A, B and C). Twenty-four hour-old
cultures of the three P. syringae strains grown on
King’s B medium were suspended in sterile distilled
water and the concentration adjusted to approx. 108
CFU/ml (OD600 = 0.3). After surface sterilization of
the vegetative shoots of seedlings, a 1–2 mm deep
hole was made into the vegetative shoots of seed-
lings by puncturing the cortex and phloem using a
sharp scalpel. A hypodermic syringe was then used
to inject 10 μl of a standardized bacterial suspension
into each of the three upper internodes of two sep-
arate shoots on each tree. Each bacterial strain was
injected into three trees of each cultivar, resulting in
six replicates for each isolate. Control shoots were
treated with sterilized water. All inoculated sites
were covered with parafilm. Lesion lengths were
measured after four weeks. The identity of re-
isolated bacterial isolates from branch cankers that
had formed was verified by oxidase, GATTa, and
HR tests. The pathogenicity trial on the cherry trees
was conducted twice.
Statistical analyses
The cherry tree inoculation data were analysed by
one-way analysis of variance (ANOVA). Mean sep-
arations were performed by Tukey’s test using R
software to determine if there was a significant
difference in pathogenicity between the respective
P. syringae strains and susceptibility of different
cherry tree cultivars. Differences at p ≤ 0.01 were
considered significant.
Eur J Plant Pathol (2018) 151:427–438
Results
Surveillance and sampling
Trees showing symptoms characteristic of bacterial can-
ker were found on all the cherry farms surveyed. These
symptoms included leaf spots, branch and stem cankers
and die-back. No blossom blast or disease symptoms on
cherry fruits were observed. The highest prevalence of
bacterial disease symptoms was in the Western Cape
with the most conspicuous symptom being branch can-
kers (Fig. 3). Cankers in all provinces were associated
with gummosis except for one farm in the Free State
where the cherry trees had dry cankers. The latter was
occasionally associated with fungal fruiting bodies.
Bacterial isolates (31) were obtained from only 5% of
620 samples that were collected and only from three
farms (farm A, B and C) in the Western Cape Province.
The isolates were obtained from different plant parts and
from a variety of cultivars (Table 4).
Bacterial identification
Phenotypic testing
The 31 bacterial strains from the current study tested
positive for levan production, gave a positive HR
reaction on tobacco and were negative for oxidase
and arginine dihydrolase and potato rot. This indicat-
ed that they were all P. syringae. The carbon utiliza-
tion tests showed that they could utilize erythritol,
inositol, sorbitol, sucrose and mannitol, thus further
characterizing them as Pss (Young and Triggs 1994).
The latter identification of the current strains as Pss
Fig. 3 Cankers associated with gummosis, the most conspicuous
symptom observed on cherry farms in the Western Cape Province
433
was confirmed with GATTa tests as all strains could
liquefy gelatine (G), hydrolyse aesculin (A), but did
not have tyrosinase (T) activity nor could they utilize
tartrate (Ta). Based on biochemical tests, 31 isolates
collected in this study represented Pss. The phenotyp-
ic profile of these strains was identical to the reference
strain Pss LMG 1247T. Even though all reference
strains had the same LOPAT profile, a difference in
the GATTa tests was noted. Psm1 LMG 2222 could
not liquefy gelatine (G) nor could it hydrolyse
aesculin (A). It could, however, show tyrosinase ac-
tivity (T) and could utilize tartrate (Ta). Psm2 CFBP
3800 could liquefy gelatine (G) but was negative for
the other GATTa tests. P. cerasi LMG 28609T was
similar to Pss LMG 1247T, but could not hydrolyse
Aesculin (A).
MLSA analyses
Based on the MLSA analyses, all 31 bacterial isolates
were confirmed to be Pss. The analyses shows the
well-supported partitioning of P. syringae strains into
three phylogroups (PG) 1,2 and 3 as shown by Nowell
et al. 2016. The 31 isolates grouped into PG 2, specif-
ically with Pss B728A and Pss HRI-W7872 with
strong bootstrap support. There was no correlation
between the strains and host (cultivar, host origin or
plant organ) but a correlation to geographic origin
could be observed. The 31 strains formed two groups:
fourteen identical isolates obtained from farm A and B
grouped together (group 1), while 17 identical isolates
obtained from farm C grouped together (group 2).
Since the strains from each group (group 1 and group
2) were identical, three reference strains from each
group (Pss 38, 82 and 83 from group 2 and Pss 56,
126 and 127 from group 1) were included in the
phylogenetic tree (Fig. 4).
Pathogenicity testing on P. avium shoots
Thirty days after inoculation, sunken, black lesions
developed from the inoculation site on succulent
shoots of cherry trees (Fig. 5). No symptoms devel-
oped on shoots treated with sterile water. There was a
significant difference (p ≤ 0.01) in pathogenicity be-
tween Pss 38, 190 and 154, the latter being more
pathogenic than the other two strains on the cultivar
Bing in both trials. There was a significant difference
in susceptibility of the three cherry tree cultivars to the
434 Eur J Plant Pathol (2018) 151:427–438

Table 4 Cherry tree cultivars

from which Pseudomonas Source of isolates
syringae pv. syringae was isolat-
ed. All isolates were obtained Sampling site Cultivar Leaves Stems Branches Number of isolates
from three sweet cherry farms
(farm A, B and C) located in the Farm A Royal Dawn 0 0 3 3
Western Cape Province Lapins 1 1 0 2
Subtotal 1 1 3 5
Farm B Bing 0 3 4 7
Royal Dawn 0 0 1 1
Van 0 1 0 1
Subtotal 0 4 5 9
Farm C Royal Dawn 1 0 2 3
Sweet heart 0 1 6 7
Star King 2 1 1 4
Stella 0 0 3 3
Subtotal 3 2 12 17
Total 4 7 20 31

Pss strains. The cultivar Bing was significantly more Discussion

resistant than the Giant Heidelfinger and Stella culti-
vars but there was no significant difference in suscep- Pseudomonas syringae is an economically important
tibility between Giant Heidelfinger and Stella cultivar pathogen of a wide range of crops including both stone
in both trials (Fig. 6). and pome fruit (Sholberg and Quamme 1999;

Fig. 4 Concatenated neighbour-

joining (NJ) phylogenetic tree
based on cts, gapA, gyrB and
rpoD gene sequences showing
three representative strains of the
14 strains isolated from farm A
and B (group1: Pseudomonas
syringae pv. syringae 38, 82 and
83) and three representative
strains from the 17 strains isolated
from farm C (group 2:
Pseudomonas syringae pv.
syringae 56, 154 and 190). All
strains group together with
Pseudomonas syringae pv.
syringae B728A and HRI-W7872
(Phylogroup 2)
Eur J Plant Pathol (2018) 151:427–438
Fig. 5 Pathogenicity trials with Pseudomonas syringae pv.
syringae strains on Stella cherry tree shoots. Sunken, black lesions
that developed from inoculation site could be observed on succu-
lent shoots 30 days post-inoculation
Lamichhane et al. 2014). As a growing industry, cherry
farming in South Africa can ill-afford losses as a result
of disease. The extreme drought and high temperatures
currently being experienced in South Africa, particularly
in the Western Cape, are exacerbating symptoms caused
by opportunistic pathogens, such as Pss. This has led to
an increased interest in the disease by the stone fruit tree
industry.
The last study of bacterial canker on cherry trees
in South Africa was conducted more than thirty
Fig. 6 Difference in canker
length of cherry tree cultivars
30 days post-inoculation with
Pseudomonas syringae pv.
syringae strains. Pseudomonas
syringae pv. syringae 154 was
significantly more pathogenic
(p ≤ 0.01) on the Bing cultivar
than the other two strains and a
significant difference (p ≤ 0.01) in
cultivar susceptibility was also
observed, with Bing being the
most resistant in both experiments
435
years ago at a time when bacterial pathogens were
predominantly characterized by biochemical testing.
Thus, the present study served to identify
P. syringae associated with bacterial canker in cher-
ry production areas of South Africa using both bio-
chemical as well as modern molecular typing
methods. The failure in this study to detect Psm
was interesting. The reasons for this result are varied
and may be due to the time of the year and differ-
ences in cultivars planted in 1980’s compared to the
current situation, and the modern technology now
available to accurately identify bacteria.
The lack of any P. syringae isolates from the Free
State, Gauteng and Mpumalanga could be attributed
to environmental factors, including the prevailing dry
environmental conditions at the time of sampling.
Alternatively, it could mean that the cause of the
observed symptoms of bacterial canker was not due
to P. syringae. Symptoms of bacterial canker can
resemble those caused by other pathogens such as:
Leucostoma, Monilinia, Prunus necrotic ring spot
virus (PNRSV) or Blumeriella jaapii (Annesi et al.
1997; Barakat and Johnson 1997; Casals et al. 2015;
436
Chandel et al. 2011). The difficulty in isolation of Pss
(isolation success of 5%) corresponds to Vicente et al.
(2004), who had an isolation success of 18%. It is
speculated that the latter could possibly have been
due to the time of sampling. In the current study, it
was probably due to the method of isolation that was
used. Crushing diseased woody tissue with a mortar
and pestle in a drop of sterile water followed by
streaking on to agar as performed by Balaž et al.
(2016) would probably have yielded a higher isolation
rate.
The MLSA provided an accurate approach for phy-
logenetic affiliation of the 31 Pss which grouped into
two clades. The geographic separation between the
farms where the respective isolates were obtained might
provide a reason for the existence of two separate groups
within the Pss strains from this study. Farm A and B are
situated next to each other in an area which experiences
lower temperatures than farm C which is 22 km further
away. Even though there are two separate groups within
the Pss isolates, the analyses showed that the strains are
highly similar. This is interesting as it is known that Pss
is heterogenous (Abbasi et al. 2013; Kałużna et al.
2010a; Khayamie et al. 2009; Iličić et al. 2016),
attacking various hosts and is dispersed in several ways
including aerosols, water and aphids (Crosse 1966;
Morris et al. 2008; Stavrinides et al. 2009). Similar
results of homo-genetic isolates have, however, recently
been reported for Pss (Balaž et al. 2016), Psm1 and
P. cerasi (Kałużna et al. 2016b). The high level of
identity of the strains shows that the Pss isolates evolved
in a specific association with the cherry trees or could
possibly be due to pathogen spread. It could, however,
also point out a common source of inoculum. The latter
is probably the case for the South African Pss strains as
the cherry trees from which they were isolated were
obtained roughly at the same time from the same nurs-
ery. The spread of bacterial canker though nurseries has
been reported by several authors (Bultreys and Kaluzna
2010; Luz 1997; Vicente et al. 2004). Insight into the
populations structure of the current Pss strains provides
valuable information on disease epidemiology and can
also be used for breeding and resistance to the pathogen,
which is currently considered to be the most effective
and economically practical disease management strate-
gy (Bassi 1999).
This study shows the current status with respect to
the prevalence of bacterial canker and the causal agent,
Pss, in cherry orchards in South Africa. The situation
Eur J Plant Pathol (2018) 151:427–438
has certainly changed in the 35 years since the last
survey conducted by Roos and Hattingh (1986). Not
only has the major cherry production areas shifted, but
so have the predominant bacterial species responsible
for canker symptoms on cherry trees and the cultivars
planted. Roos and Hattingh (1986, 1987a) reported the
prevalence of Psm1 in the Free State Province and
occurrence of Pss, Psm1 and Psm2 in the Western Cape.
The current study, however, showed only the presence
of a homogenous Pss population in the Western Cape,
which is becoming the major cherry producing region of
South Africa. In the 1980s the cultivars BCorum^ and
BEmperor Francis^ were well established (Roos and
Hattingh 1986) whereas today neither cultivar is grown
and new cultivars such as BBlack Star^ occur. Further
studies are needed to clarify whether all symptoms
observed in this study are in fact as a result of bacterial
canker, or if other causal agents are of importance. This
is critical for implementing effective management and
the fine-tuning management strategies to ensure higher
production from cherry farming.
Acknowledgements The Horticultural Knowledge Group
(HORTGRO) and National Research Foundation (NRF) are ac-
knowledged for funding this research. In addition, the cherry
farmers are acknowledged for access to their farms and informa-
tion provided.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
Human participants and animal studies No humans or ani-
mals were involved in the execution of this research. All authors
have consented to the submission of this manuscript to EJPP.
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